The altered activity of P53 signaling pathway by STK11 gene mutations and its cancer phenotype in Peutz-Jeghers syndrome

Peutz-Jeghers syndrome (PJS; OMIM #175200) is an autosomal dominant disorder characterised by mucocutaneous melanin pigmentation (MP), gastrointestinal(GI) hamartomatous polyposis, and an increased risk for the development of various neoplasms [1, 2], which is rare with a low incidence of 1/50,000 [3]. The cumulative lifetime risk is 20, 43, 71, and 89% at ages of 40, 50, 60 and 65 years, respectively [4].

Germline mutations in the serine–threonine kinase 11 (STK11) gene on chromosome 19p13.3 were identified as a major genetic cause of PJS in 1998 [5, 6]. This gene is divided into 9 coding exons that encode a 433 amino-acid protein, which acts as a tumor suppressor. The STK11 protein is mainly comprised of 3 major domains: an N-terminal regulatory domain, a catalytic kinase domain, and a C terminal regulatory domain [7]. Amino acids 49–309 of STK11 are the catalytic kinase domain, which can form a complex with STRAD and scaffold protein 25 (MO25) to maintain kinase activation [8]. Several studies have described a role of STK11 in cell cycle arrest [9], P53 mediated apoptosis [10], Wnt signaling [11, 12], TGF-β signaling [13], Ras induced cell transformation [14], and cell polarity [15–18].

P53, the most widely studied tumor suppressor gene, regulates of the expression of various genes that are related to cell cycle arrest, DNA damage repair and apoptosis [19], and furtherly controls the cell proliferation and apoptosis. P53 gene mutations have been found in about 50% of human cancers, suggesting the important role of P53 inactivation in tumorigenesis [20]. STK11 has been reported to play a critical role in P53-mediated cell apoptosis [10]. STK11^+/−/P53^+/− mice showed a dramatically reduced life span and increased tumor incidence compared to the mice with either STK11 or P53 single gene knockout, indicating that P53 and STK11 gene mutations cooperate in tumor progression [21].

In this study, we identified mutations in STK11 gene from Chinese PJS probands, and observed the changes in P53 activity brought by different STK11 mutants and their association with the canceration in PJS.

During the period investigated, 154 PJS patients were diagnosed in our department, among whom, 89 declined to participant in mutation test, and 26 had inadequate information. Finally, 39 patients from 31 unrelated families had blood samples collected and received mutation detection. A total of 17 disease-specific mutations in STK11 were detected in 31 probands. Since 3 mutations concerning splicing site (c.290 + 1G > A, c.734-1G > A, c.863-8_870del16bp) could not be investigated by reporter gene assay, and one mutation (c.426_448del23bp) was hard to be generated by site-directed mutagenesis, there were 13 mutants involved in the final analysis (Additional file 2: Figure S1). Expression vectors of the wild-type and the 13 mutant STK11 gene were constructed for function analysis, and the corresponding PJS families and individuals were included in analysis.

Clinical features

As shown in Table 1, the 13 probands from unrelated families were 7 familial and 6 sporadic patients, and their native places were various in China (Additional file 1: Table S2). The median age of diagnosis, first polypectomy and at present was 25, 28 and 33 years. Seven of these families (PJS03, 05, 06, 07, 09, 10 and 12) showed autosomal dominant pattern and the remaining 6 cases (PJS01, 02, 04, 08, 11 and 13) are sporadic (Fig. 1a). MP on the lips, buccal mucosa, and digits were observed at all patients (Fig. 1b). Twelve probands in these families underwent laparotomy or polypectomy due to intussusception or intestinal obstruction (Fig. 1c and d). Three probands of these families (PJS03, 06 and 11) in this study had developed colon cancer before 40 years old (Fig. 1e), and cancer history existed in 3 families (PJS06, 09 and 12). Detail information is showed in Table 2.

Characteristics		Number (percentage)
Probands with mutation test		31
Mutation detected		17	54.8%
Probands enrolled in the luciferase analysis		13
Agea Median (range)	D	25 (12–37) years
	F	28 (11–39) years
	P	33 (24–51) years
Gender
Male		7	51.8%
Female		6	48.2%
Family history
Familial		7	51.8%
Sporadic		6	48.2%
Cancer history		5	38.5%

^a D, diagnosis; F, first polypectomy or laparotomy; P, present. There were 12 probands having polypectomy

ID	FHa	Sexb	Agec			Polyp location	Int	Mutation	Exon	Function domain	Effect	Function Prediction		ExAC allele frequency	P53 activity	Index cancer	Cancers in family
			D	F	P							PolyPhen-2	SIFT
PJS01	S	M	12	11	24	SB, C	Yes	c.56C > G	1	N	p.S19 W	0.663	0.00	0	Normal
PJS02	S	F	15	15	29	SB	Yes	c.193G > T	1	I	p.E65a	NA	NA	0	Decreased
PJS03	F	F	31	27	33	SB, C	Yes	c.250A > T	1	II	p.K84a	NA	NA	0	Decreased	colon
PJS04	S	F	17	12	28	SB	Yes	c.354C > G	2	IV	p.Y118a	NA	NA	0	Decreased
PJS05	F	F	23	23	27	SB, C	Yes	c.842delC	6	XI	p.P281Rfsa6	NA	NA	0	Decreased
PJS06	F	M	21	21	45	SB, C	Yes	c.843insC	6	XI	p.P281Pfsa4	NA	NA	0	Decreased	colon	lung, lymphoma
PJS07	F	M	32	32	38	SB, C	Yes	c.855delG	6	XI	p.L285Lfsa2	NA	NA	0	Decreased
PJS08	S	F	31	15	35	G, SB, C	Yes	c.862G > A	6	XI	p.G288S	0.0.573	0.10	0	Normal
PJS09	F	M	30	30	51	SB, C	No	c.911G > C	7	XI	p.R304P	0.0.987	0.05	0	Normal		stomach
PJS10	F	M	24	39	43	SB, C	Yes	c.924G > A	8	XI	p.W308a	NA	NA	0	Normal
PJS11	S	M	37	37	47	C	Yes	c.962_963delCC	8	C	p.P321Hfsa38	NA	NA	0	Normal	colon
PJS12	F	F	12	12	29	G, SB, C	Yes	c.1062C > G	8	C	p.F354 L	0.022	0.36	0.005757	Normal		colon
PJS13	S	M	24	23	30	G	Yes	c.1225C > T	9	C	p.R409W	0.549	0.00	0.000147	Normal

^aFamily history. S = sporadic, F = familial. ^b M = male, F = female. ^cD, diagnosis; F, first polypectomy or laparotomy; P, present. If a patient is dead, the present age with a underline refers to the age of death. G = stomach, SB = small bowel, C = colon. Int = intussusception. NA = not available

Mutation analysis

Sequencing analysis identified 13 mutations of STK11 gene in the 13 probands which were included in the function analysis (Table 2, Fig. 2a). Most of the mutations have been previously reported [25, 27–29] except for c.924G > A (Fig. 3). The 13 mutations included 8 truncating mutations (4 frameshift and 4 nonsense) and 5 missense mutations. Regarding frameshift mutations, those of PJS05, 06 and 07 (p.P281Rfs*6, p.P281Pfs*4 and p.L285Lfs*2) led to a partial loss of the kinase domain and a completed loss of the C-terminal, and the one in PJS11 (p.P321Hfs*38) led to a partial loss of the α-helix of the C-terminus. PJS02, 03, 04 and 10 possessed nonsense mutations of STK11 that resulted in the production of truncated proteins (p.E65*, p.K84*, p.Y118* and p.W308*) (Additional file 2: Figure S2A). Among the PJS cases owning the 13 mutations, 7 were familial and 6 were sporadic, and 5 of them were associated with cancer history. Tested in 100 chromosomes from control individuals, 16 of the detected mutations were not discovered and only c.1062C > G was detected in 5/100 controls. c.1062C > G and c.1225C > T were recorded in ExAC database with allele frequencies of 0.005757 and 0.000147, and all other mutations were not recorded (Table 2).

Peutz-Jeghers syndrome (PJS) has various manifestations related to GI polyps, such as abdominal pain, rectal blood loss, chronic anemia, prolapsed rectal polyp, bowel obstruction and clinical intussusception [30–32]. Beyond these symptoms, PJS patients also have an increased risk of cancer at multiple sites, including the GI tract, breast, ovary, testis, and lung [33]. As the only validated and the major pathogenic gene in PJS, STK11 is involved in cell cycle regulation and apoptosis, whose abnormality can induce and promote tumorigenesis. It has been proven that STK11-mediated cell cycle regulation has been shown to be regulated via P53-dependent and P53-independent mechanisms [34–39]. STK11 can directly interact with and phosphorylate P53, and to mediate P53-induced apoptosis [40]. What’s more, PJS-associated STK11 mutants can diminish P53 activity [41].

In our study, we investigated the effect of STK11 mutations on the transcriptional activity of P53, and we found that six of them inhibited the activity significantly compared with the wild-type. All of the 6 mutations resulted in protein truncation, and all the truncated protein lost partial kinase domain and completed C-terminal. The other seven mutations which didn’t significantly decrease P53 activity located in the margin of or outside the kinase domain, and they caused only single amino acid change in the margin of or outside the kinase domain or loss of C-terminal regulatory domain. So we suspected the main part of the kinase domain or some key points within it was key for STK11 depended P53 activity rather than the boundary of it or regulatory parts, and truncating mutations were easier to cause the change since they usually resulted in large-scale loss of the amino acid residues. We also encountered three splice site mutations, which cannot be investigated by the luciferase assay. Since splicing errors usually cause exon skipping or abnormal mRNA processing and mRNA degradation, the large-scale changes may also result in P53 activity decrease. But due to the limitation of patient number, the assumption cannot be justified at the present.

As to the association between P53 activity reduction and cancerogenesis, there is no significance under statistical analysis. One of the reasons is the limited number of cases involved in analysis, just like another two analyses (sex and family history) without statistical significance. When analysed one by one, we can discover some implications from them. Early onset is a distinguishing feature of inherited cancer, and among our cases, there are cancers patients younger than 40 in PJS03 and 06 whose P53 activity is reduced by their mutations. While in PJS09, 11 and 12, cancer patients are older than 45, which means they are more like sporadic ones. Though there is a death case of youth in PJS12, it is not a certain case of cancer. More important, the mutation c.1062C > G is now regarded as a benign variation according to guideline from American College of Medical Genetics and Genomics (ACMG) [42, 43]. So most likely there is a large scale deletion rather than point mutation like c.1062C > G which is to blame. Another possibility is that P53 pathway is not the key one taking part in these three PJS families’ cancerogenesis, and some non-P53 pathways leads to the occurrence of tumors.

There are still four families without cancer diagnosed, whose mutations were tested to impact P53 activity. The four mutation carriers are still young (29, 28, 27 and 38 years), so it is important to carry out a more rigorous and comprehensive follow-up as cancer prevention before any advanced methods come up. As to the other four PJS families carrying STK11 mutations without significant P53 activity change, routine surveillance is still necessary to keep them uneventful. The recommendations for PJS management produced by Mallorca conference 2007 is a comprehensive and widely accepted one, which we can refer to [44, 45].

There are several important limitations to note. First, here we try to elucidate the pathways of tumorigenesis in PJS by investigating the P53 activity change caused by STK11 mutations, but the mutations are not evenly distributed within the coding sequence and the limited number of cases involved largely limits the results. Second, quite a few of STK11 mutations in PJS are large fragment deletion, so it is necessary to perform test like multiplex ligation–dependent probe amplification (MLPA) assay to make a more comprehensive analysis in the future. Finally, this is a single center study, and the data here may not be the whole picture of PJS in China. It will be very helpful if there is a national database of PJS patients.

The affected P53 activity caused by STK11 mutations in PJS patients is significantly associated with protein truncation, while cancer risk in PJS can be elevated through pathways rather than P53 pathway. P53 activity test is probably a useful supporting method to predict cancer risk in PJS, which could be helpful in clinical practice.